Kim et al. J Hematol Oncol (2021) 14:43 https://doi.org/10.1186/s13045-021-01058-6
RESEARCH
Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapyHye‑Ran Kim1,2,3†, Jeong‑Su Park1,2†, Jin‑Hwa Park4, Fatima Yasmin1,2, Chang‑Hyun Kim1,2, Se Kyu Oh4, Ik‑Joo Chung5 and Chang‑Duk Jun1,2*
Abstract
Background: Transgelin‑2 is a 22 kDa actin‑binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin‑2 in dendritic cell (DC) immunity. Further, we inves‑tigated whether the non‑viral transduction of cell‑permeable transgelin‑2 peptide potentially enhance DC‑based cancer immunotherapy.
Methods: To understand the functions of transgelin‑2 in DCs, we utilized bone marrow‑derived DCs (BMDCs) puri‑fied from transgelin‑2 knockout (Tagln2−/−) mice. To observe the dynamic cellular mechanism of transgelin‑2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T–DC interaction in vivo, we used intravital two‑photon microscopy. For the solid and metastasis tumor models, OVA+ B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell‑permeable, de‑ubiquitinated recombinant transgelin‑2 was purified from Escherichia coli and applied for DC‑based adoptive immunotherapy.
Results: We found that transgelin‑2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC‑based immunity. Although Tagln2−/− BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen‑specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA+ B16F10 melanoma cells in mice. Tagln2−/− BMDCs had defects in filopodia‑like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remod‑eling in vitro and formed short, unstable contacts with cognate CD4+ T cells in vivo. Strikingly, non‑viral transduction of cell‑permeable, de‑ubiquitinated recombinant transgelin‑2 potentiated DC functions to suppress tumor growth and metastasis.
Conclusion: This work demonstrates that transgelin‑2 is an essential protein for both cancer and immunity. There‑fore, transgelin‑2 can act as a double‑edged sword depending on how we apply this protein to cancer therapy.
*Correspondence: [email protected]†Hye‑Ran Kim and Jeong‑Su Park contributed equally to this work1 School of Life Sciences, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi‑ro, Gwangju 61005, KoreaFull list of author information is available at the end of the article
Page 2 of 23Kim et al. J Hematol Oncol (2021) 14:43
IntroductionDendritic cells (DCs) are professional antigen-presenting cells that survey tissues for foreign antigens [1, 2]. Fol-lowing an encounter with a foreign antigen, DCs are acti-vated in a process involving the capture and processing of the antigen, expression of lymphocyte co-stimulatory molecules, migration to lymphoid tissues like the spleen and lymph node for completion of their maturation, and secretion of cytokines to initiate the adaptive immune response [3, 4]. During maturation and migration to the lymph node, DCs undergo global rearrangements of the actin cytoskeleton, which are mediated through specific temporal and spatial actions of actin-binding or regula-tory proteins [1]. Rac1/2, a small G-protein responsible for ruffling movements, is essential for the interaction between DCs and T cells [5, 6]. The formin mDia1 is essential for DC adhesion, migration, and sustained interaction with T cells [7]. Wiskott–Aldrich syndrome protein, a molecule that controls Arp2/3-dependent actin polymerization, is required for the formation of the immunological synapse (IS) and DC migration [8–10]. The cortactin HS1 is necessary for organizing the podo-some array and is primarily required for directional per-sistence of migrating DCs [11, 12]. However, none of these actin regulators is specific for DC functions as they are ubiquitously expressed and function in most mam-malian cells [1]. Thus, the discovery of a DC-specific actin regulatory protein would help us understand how DC immunity is linked to dynamic actin remodeling at a fundamental level.
Transgelin-2, a 22-kDa actin-binding protein, is one of three transgelin family members characterized by their actin cross-linking and gelling properties [13]. Although the topic is still debated, transgelin-2 has been impli-cated in tumorigenesis and cancer development [14]. Indeed, its upregulation is correlated with the clinical stage, tumor size, and invasion in a wide spectrum of cancers [15]. We previously found that transgelin-2 is also expressed in lymphocytes and functions to stabilize the immunological synapse, thereby enhancing T cell activation [16, 17]. It is also involved in filopodium ini-tiation and/or elongation presumably by interfering with the interaction between the Arp2/3 complex and actin [18], which may drive the enhanced phagocytic behav-ior of macrophages toward invading bacteria [19]. Taken
together, these results suggest that transgelin-2 is not only important for tumorigenesis and cancer progression but is also essential for immune functions.
In the present study, we observed that although transgelin-2 is not at all or only minimally expressed in immature BMDCs, it is dramatically expressed dur-ing granulocyte–macrophage colony-stimulating fac-tor (GM-CSF)- or FMS-like tyrosine kinase 3 ligand (Flt3L)-induced maturation and lipopolysaccharide (LPS) activation. This suggests that transgelin-2 may play a role during DC maturation or DC-mediated priming of antigen-specific T cells. In support of this idea, previous reports demonstrated that the actin bundling protein fas-cin is induced upon DC maturation and involved in the antigen presentation activities of mature DCs [20, 21]. Why would DCs require the expression of an actin bun-dling protein specific to DCs? Since the main functions of DCs, which distinguish them from other cells, are to continuously capture, deliver, and process antigens and present them to T cells [22], mature DCs may require actin regulatory proteins optimized for DC functions. In addition, this suggests that these proteins are not redun-dant and that each may have distinctive roles for mature DC functions. Here, we investigated the subcellular local-ization and functions of transgelin-2 in BMDCs. Two-photon microscopy was utilized to monitor the in vivo migration of BMDCs, as well as their dynamic interac-tion with T cells. BMDCs with genetic ablation of Tagln2 (Tagln2−/−) exhibited significant defects in homing to the draining lymph node and priming of antigen-specific T cells for clonal expansion and cytokine production. Sur-prisingly, exogenous introduction of a cell-permeable and ubiquitination site-mutated (K78R) recombinant transgelin-2 (dU-TG2P) into BMDCs significantly poten-tiated tumor regression in vivo, suggesting a potential use for transgelin-2 peptides in DC-mediated anticancer therapy. In summary, our findings indicate that transge-lin-2 positively regulates DC-mediated adaptive immune responses.
ResultsTagln2‑knockout (Tagln2−/−) DCs do not optimally control B16F10 tumor metastasis and growth in micePreviously, we reported that transgelin-2 is highly expressed in immune-related tissues, such as the
Engineering and clinical application of this protein may unveil a new era in DC‑based cancer immunotherapy. Our findings indicate that cell‑permeable transgelin‑2 have a potential clinical value as a cancer immunotherapy based on DCs.
Page 3 of 23Kim et al. J Hematol Oncol (2021) 14:43
thymus, spleen, and LNs [16]. Transgelin-2 is also dom-inantly expressed in lymphocytes and plays an impor-tant role in stabilizing the IS, thereby enhancing T cell-mediated immune responses [16, 17, 23]. In addi-tion, we found that transgelin-2 is physically associ-ated with the integrin lymphocyte function-associated antigen-1 (LFA-1), which enhances the adhesion of cytotoxic T cells to intercellular adhesion molecule-1 (ICAM-1)-positive tumor target cells, such as E0771 cells, but not ICAM-1-negative B16F10 cells [17]. In accordance with the previous results, overexpression of transgelin-2 (TG2) in OTI T cells did not enhance T cell adhesion to ovalbumin (OVA)-pulsed B16F10 target cells in vitro and showed only a mild effect on B16F10 tumor regression in vivo (Fig. 1a, b). Interest-ingly, however, we observed that whole-body transge-lin-2-KO (Tagln2−/−) mice, as compared to wild-type (WT) mice, developed rapid B16F10-derived tumor growth, resulting in increased tumor weights and sizes (Fig. 1c). Kaplan–Meier survival studies 40 days after tumor injection showed that WT mice had a median survival time of 35 days. However, Tagln2−/− mice had a median survival time of only 28 days (Fig. 1c). These results suggest that cell types other than T cells can participate in tumor regression independently of the LFA-1/ICAM-1 interaction between cytotoxic T cells and tumor target cells.
Since DCs play a major role in processing and pre-senting antigen peptides to antigen-specific T cells, we next asked whether DCs express transgelin-2 or other isotypes, such as transgelin-1 and -3. Interestingly, immature DCs isolated from bone marrow (BM) did not express transgelin family members (Fig. 1d). How-ever, transgelin-2 was highly expressed during GM-CSF-induced differentiation (Fig. 1d). The expression of transgelin-2 was also induced in fully differentiated
BMDCs in response to LPS (Fig. 1e), suggesting a spe-cific role of transgelin-2 in mature BMDCs.
To understand the significance of transgelin-2 expres-sion in mature DCs, we used mature BMDCs from WT (Tagln2+/+) and Tagln2−/− mice (Fig. 1f ), which were previously generated in our laboratory [16]. To determine whether transgelin-2 regulates the functions of BMDCs, OVA257–264-pulsed WT or transgelin-2-knockout (KO) BMDCs were i.v. injected into mice, thereby generating cytotoxic T cell clones against the OVA257–264 peptide in vivo (Fig. 1g). At 7 days after DC injection, the mice were i.v. injected with OVA+B16F10 melanoma cells. The metastatic colonies were evaluated at day 14, and a sig-nificant increase in metastatic nodules was observed in mice with adoptively transferred OVA257–264-pulsed Tagln2−/− BMDCs compared to OVA257–264-pulsed WT BMDCs (Fig. 1h). For the solid tumor model, OVA+B16F10 cells were implanted into the mammary fat pads of C57BL/6 female mice previously injected with WT BMDCs or Tagln2−/− BMDCs. In accordance with the metastatic model, Tagln2−/− BMDCs could not opti-mally control tumor growth in mice (Fig. 1i). Kaplan–Meier survival studies showed that mice injected with OVA257–264-pulsed WT BMDCs had a median sur-vival time of 40 days. However, mice adoptively trans-ferred with OVA257–264-pulsed Tagln2−/− BMDCs had a median survival time of only 34 days similar to PBS control group (Fig. 1j). Further, we observed a significant reduction in CD8+ tumor-infiltrating lymphocytes (TILs) in the B16F10 tumors from Tagln2−/− DC-injected mice (WT vs. KO, 9.8 ± 1.5 vs. 1.8 ± 1.1) (Fig. 1k). In addition, the tumor-infiltrating CD8+ T cells from Tagln2−/− DC-injected mice expressed a lower level of IFNγ than that in WT DC-injected mice (Fig. 1l). Among various DC subsets, conventional type 1 DCs (cDC1s) are the main cellular source of IL-12, a fundamental cytokine for anti-cancer CD8+ CTL activation, and the super promising
(See figure on next page.)Fig. 1 Tagln2−/− BMDCs do not optimally control B16F10 tumor metastasis and growth in mice. a Effect of transgelin‑2 expression in T cells for adhesion to B16F10 tumor cells. Empty vector (EV)‑ or transgelin‑2 (TG2)‑expressing OTI CD8+ T cells were co‑incubated with B16F10 in the presence or absence of OVA peptide for 2 h, and conjugates were analyzed by flow cytometry. b Transgelin‑2 in T cells showed a minimal effect on B16F10 tumor growth in mice. PBS (none), WT (OTI‑T), KO (Tagln2−/− OTI‑T), or transgelin‑2‑overexpressing T (TG2OE OTI‑T) cells were adoptively injected into C57BL/6 mice after OVA+B16F10 inoculation. B16F10 tumor weights were measured at day 25 post‑implantation (n = 7). c Gross images of 8‑day‑old OVA+B16F10 melanoma after s.c. inoculation (3 × 105) in C57BL/6 WT or Tagln2−/− mice. Tumor weights and sizes were measured at day 8 post‑implantation (n = 7). The survival rates of tumor‑bearing mice post‑implantation are shown. d Expression of transgelins during GM‑CSF‑induced differentiation. Freshly isolated BM cells were treated with 20 ng/mL GM‑CSF and harvested at the indicated days for western blot. e Expression of transgelins in differentiated DCs after LPS (200 ng/mL) stimulation. f Transgelin‑2 expression in DCs from WT or Tagln2−/− mice. Results are representative of three independent experiments (d–f). g Schematic diagram of solid and metastatic tumor models. h, i Gross images of OVA+B16F10 lung metastasis and solid tumors. C57BL/6 mice were injected i.v. with media alone, WT DCs, or Tagln2−/− DCs. After 7 days, the mice were i.v. (h) or s.c. (i) injected with OVA+B16F10 cells. The metastatic nodules (h) and tumor weights and sizes (i) were quantified after 8 days. j The survival rates of tumor‑bearing mice post‑implantation are shown. Data are representative for nine mice in each group. k, l Tumors from I were dissected, and the populations of infiltrated CD3+CD8+ T cells (k) and the amount of intracellular IFN‑γ in TILs were determined by flow cytometry (l). All data from a–l represent the mean of three experiments ± SEM. *P < 0.05; **P < 0.001; ***P < 0.0001
Page 4 of 23Kim et al. J Hematol Oncol (2021) 14:43
TG2
β-actin
0
WT
0 24+ LPS (h)
KO
24
22
45
TG2
β-actin
0 6 9 12Hours
+ LPS
24
22
45
TG1
TG3
22
22
20 1 3 64 5Days
+ GM-CSF
22
45
22
22
TG2
β-actin
TG1
TG3
TW
OK
WT KO W
T KO
Mature-BMDC
Tagln2 gene
Sur
viva
l pro
babi
lity
(%)
00
25
50
75
100
Days
WT
25 30 35 40 45
WT
TG2OE
Tum
or w
eigh
t (g)
0
0.1
0.2
0.3
0.4
TG2KO
OT-I T
None
0.5
0.6
0
2.5
5
7.5
10%
Con
juga
tes
EV-OTI TTG2-OTI T
- OVA
+ OVA
B16F10 B16F10
B16F10(for metastasis)
i.v. injection (OVA257-264-pulsed wt-DC or KO-DC)
Metastasis andsolid tumor models
0.66
84.4
102
103
104
0
0.34
94.4
0 102 103 104
WT KO
CD-T
WC
D-O
Kaide
M *seludon fo .o
N
KOW
TMOCK
Tagln2 gene
0 102 103 104
B16F10(Solid tumor)
KO-DC
WT-D
C
Media
MOCKW
T KO0.0
0.4
0.8
1.2
1.6 p = 0.0053
0.0
0.1
0.2
0.3
0.4
0.5
MOCK WT KO
p = 0.0032
Sur
viva
l pro
babi
lity
(%)
00
25
50
75
100
Days25 30 35 40 45
MOCK
*
KOW
T
NFI fo %
γLIT 8
DC fo +
a
d
g
ij
k
l
h
e f
b c
Page 5 of 23Kim et al. J Hematol Oncol (2021) 14:43
DC subset for induction of anti-tumor immunity due to their superior capacity to uptake dying or dead cell mate-rials and to process tumor-associated antigens for cross-presentation [24–26]. We therefore determined whether transgelin-2 is also expressed in Flt3L-induced cDC1s. To this end, BM-isolated immature DCs were treated with Flt3 ligand for 9 days, and analyzed the expression of surface markers and transgelin-2 (Additional file 1: Fig. S1A and B). We found that transgelin-2 was also dramati-cally induced during Flt3L-induced cDC1 differentiation, while transgelin-2-KO followed the normal differen-tiation patterns as judged by the expressions of CD103, CD24, and XCR1 (Additional file 1: Fig. S1A and B). We next evaluated the antitumor activity of Tagln2−/− cDC1s against s.c. injected OVA+B16F10 cells. Similar to the GM-CSF-induced BMDCs, Tagln2−/− cDC1s were not able to optimally control tumor growth in mice (Addi-tional file 1: Fig. S1C), suggesting that transgelin-2 is also important for the function of cDC1s to facilitate optimal antigen presentation.
Transgelin‑2 is essential for global actin rearrangements in activated DCsTo investigate the mechanism by which transge-lin-2 affects the function of DCs, we analyzed mature BMDCs obtained from Tagln2−/− mice and compared them with WT BMDCs in terms of their morphol-ogy, actin dynamics, and signaling. Morphologically, although there were no gross differences between WT and KO mice, Tagln2−/− BMDCs exhibited an absence of filopodia-like membrane protrusions, as determined by scanning electron microscopy (SEM) (Fig. 2a, yel-low arrowheads). To understand this morphological phenotype, LPS-stimulated WT BMDCs were stained for F-actin and transgelin-2 and visualized by confo-cal microscopy. As shown in Fig. 2b, transgelin-2 was specifically localized at the leading edge of lamellipo-dia, where filopodia are formed from the pre-existing meshwork of Arp2/3 complex-branched filaments. In addition, transgelin-2 co-localized with F-actin in the actin-rich cores of podosomes, which were surrounded by vinculin in the stereotypical podosome organization
(Fig. 2b, right). Podosomes are characteristics of cells in the myeloid lineage, including DCs, macrophages, and osteoclasts, which can degrade extracellular matri-ces and play a role in the migration of DCs through tissues [27]. Thus, the localization of transgelin-2 in filopodia tips, as well as in podosomes, demonstrated that transgelin-2 is involved in the membrane protru-sions that support DC migration and interactions with T cells for proper antigen presentation. To this end, we next assessed podosome formation and spreading dur-ing DC activation in response to LPS and fibronectin (Fn), respectively.
DCs have been shown to exhibit transient podosome loss at approximately 20 min after their activation with LPS and then recover them again after 2 h [27, 28]. Like-wise, in our study, BMDCs from both WT and KO mice showed a substantial reduction in the number of cells with podosomes after 30 min of stimulation with LPS. However, podosomes were significantly slower to reform in Tagln2−/− BMDCs after 2 h (Fig. 2c). Next, since the interaction of BMDCs with extracellular matrix (ECM) proteins, such as Fn, induces a dynamic cytoskeletal rear-rangement driven by actin polymerization, thereby medi-ating cell adhesion and migration through integrins [28, 29], we evaluated the effects of the transgelin-2 KO on DC spreading and actin polymerization. Compared to WT BMDCs, Tagln2−/− BMDCs exhibited reduced cell spreading and lower levels of polymerized actin when seeded on Fn (Fig. 2d). Taken together, these results strongly suggest that transgelin-2 has a specific role in DC migration by modulating filopodia and podosome formation, and cell spreading on ECM.
CCR7 is necessary for directing DCs to secondary lym-phoid nodes and eliciting an adaptive immune response [30, 31]. Furthermore, CCR7 induces actin rearrange-ments by activating Akt and Erk signaling, as well as the Gi-dependent activation of MAPK members Erk1/2, Jnk, and p38 [32]. To determine whether transgelin-2 is linked to dynamic actin signaling in mature BMDCs, we examined signaling downstream of CCR7 activation by CCL19. PI3K and its downstream effector Akt were both significantly attenuated in Tagln2−/− BMDCs. Similarly,
Fig. 2 Transgelin‑2 is essential for global actin rearrangements in activating DCs. a SEM images of WT or Tagln2−/− DCs in the resting state. b Localization of transgelin‑2 in DCs. Enlarged boxed images show the localization of transgelin‑2 in the filopodial tip at the edge of a protrusive region (left) and podosomes, along with F‑actin and vinculin (right). The fluorescence intensity profiles of each protein were analyzed using Fluoview. Scale bar, 5 μm. c Re‑assembly of podosomes in WT or Tagln2−/− DCs in response to LPS. Podosome cores were visualized by phalloidin staining, and the number of podosomes per cell was counted as described in Materials and Methods. Scale bar, 5 μm. d Effects of transgelin‑2‑deficiency on DC spreading and actin polymerization. WT or Tagln2−/− DCs were seeded on Fn‑coated coverslips and stained with phalloidin‑TRITC. Cell spreading areas were calculated using ImageJ software. F‑actin contents were analyzed by flow cytometry. Data from c and d represent the mean of three experiments ± SEM. *P < 0.05, **P < 0.001. e Determination of signaling cascade in WT or Tagln2−/− DCs in response to CCL19 (200 ng/mL) by western blot. Results are representative of three independent experiments
(See figure on next page.)
Page 6 of 23Kim et al. J Hematol Oncol (2021) 14:43
WT- LPS 30 min 2 h
KO**
80
60
100
% P
odos
ome+ c
ells
20
0
40
- LPS+ LPS (h) 0.5 2
TG2
Actin Merged
Vinculin Actin
TG2
Merged
Merged
WTKO
0 10 20 30 400
1
2
3
4
FI (x
100
0)
Actin TG2Vinculin
Are
a of
cel
l spr
eadi
ng (µ
m2 )
WT KO
0
100
200
400
300
500
WTKO
**
filopodiafilopodia tip
+ LPS
p-PI3K (p85)50 2+ CCL19 (min) 10
p-p38
p-Akt
p-Erk
20 50 2 10 20
p-PI3K (p55)
M85
42
60
44
55
42
t-Akt 60
t-p38 42
85t-PI3K (p85)
t-Erk4442
Tagln2-/- DC Tagln2+/+ DC
Filopodia tip
ActinTG2
Stress fiber
1
2
3
4
0 2 4 6 8 10
F-ac
tinco
nten
t(FI
)
Fn (10 µg/ml)(min)
** **
*
Tagln2+/+ DCTagln2-/- DC
Ventral plasma membranePodosome plaque/protrusion
Tagln2-/- DC Tagln2+/+ DC
T agln2
-/-D
C
T agln2
+/+ D
C
d
e
a
b
c
Page 7 of 23Kim et al. J Hematol Oncol (2021) 14:43
p38 and Erk signaling were also reduced in these cells (Fig. 2e).
Tagln2−/− DCs showed impaired migration into the lymph nodeReduced CCR7-mediated signaling led us to examine whether transgelin-2 deficiency was connected with the expression of chemokine receptors or adhesion mol-ecules on the surface of DCs. We found no differences in the expression levels of surface proteins between WT and KO BMDCs. However, Tagln2−/− BMDCs exhib-ited dramatic defects in migration in response to vari-ous chemokines, such as CCL3, 5, 19, 21, and SDF-1α (Fig. 3a). We further performed live time-lapse imaging to further monitor how DCs responded to stimulation with the chemokine CCL19. Although WT BMDCs read-ily spread, formed membrane protrusions, and showed a typical dendritic morphology, Tagln2−/− BMDCs were round and irregularly shaped, with a relative absence of a protrusive morphology (Fig. 3b and additional movie files show this in more detail. [See Additional files 2–5]), suggesting that the spatial and temporal regulation of the actin cytoskeleton is impaired in Tagln2−/− BMDCs.
We therefore next asked whether DC migration was also impaired in vivo in Tagln2−/− mice. To this end, we co-injected differentially labeled BMDCs from WT or Tagln2−/− mice into the footpads of normal recipient mice. Draining popliteal LNs were collected at 24 h post-injection, and the presence of migrated DCs was assessed by flow cytometry (Fig. 3c). Fixed tissues were also exam-ined under confocal microscopy (Fig. 3d). In contrast to WT BMDCs, which reached the draining LNs nor-mally, there was a significant reduction in the number of Tagln2−/− BMDCs observed in the draining LNs (Fig. 3c, d), thus demonstrating that transgelin-2 influences the migration rate of DCs in vivo.
Tagln2−/− DCs do not optimally support T cell activationAlthough enlarged draining popliteal LNs were observed in recipient OTII TCR mice adoptively transferred with OVA323–339-pulsed WT BMDCs, no significant change in LN size was seen with Tagln2−/− BMDCs (Fig. 4a). This result suggests that
antigen-specific OTII CD4+ T cells may be less pro-liferative in mice injected with Tagln2−/− BMDCs. As expected, the population of Tagln2−/− BMDCs (CD11c+ cells) that migrated into the draining LN was significantly smaller than that of WT BMDCs (Fig. 4b). Interestingly, however, the expression of co-stimulatory molecules, such as CD80 and CD40, was not altered in Tagln2−/− BMDCs in vivo, suggesting that transgelin-2 has little effect on the ability of DCs to express these co-stimulatory factors. This result led us to ask whether the migratory defects seen in Tagln2−/− BMDCs were the only driver of the reduced antitumor response (Fig. 1h, i) or whether another mechanism was also involved. To this end, we investigated the role of transgelin-2 in the direct DC-mediated T cell response in vitro. OVA323–339-pulsed BMDCs were co-cultured with OTII CD4+ T cells, and the surface expression of CD69 or CD25 on T cells was determined. Interestingly, the expression of these activation markers was significantly reduced in T cells co-cultured with Tagln2−/− BMDCs, thereby indi-cating a second role for transgelin-2 in DCs.
To understand how Tagln2−/− BMDCs attenuated T cell activation in vivo, we performed an in vitro activa-tion assay. We first determined whether transgelin-2 KO affected the differentiation of immature DCs into mature DCs. During GM-CSF-induced differentiation, Tagln2−/− BM cells followed the normal differentia-tion patterns based on the surface expression of CD11c (Fig. 5a). In addition, the expression levels of MHC class II and co-stimulatory molecules, such as CD80, CD86, and CD40, were not significantly different in either WT or Tagln2−/− BMDCs by LPS stimulation (Fig. 5b). Further, the expression levels of DC cytokines such as IL-1β and IL-12 were similar in both WT and Tagln2−/− BMDCs (Fig. 5c). By contrast, the activa-tion of OTII CD4+ T cells was significantly reduced in OVA323–339-pulsed Tagln2−/− BMDCs as determined by the expression of CD69 and CD25, and the secre-tion of IL-4, IL-2, and IFN-γ (Fig. 5d–f ). Indeed, higher levels of antigen peptide were required to produce a similar amount of cytokines in the Tagln2−/− BMDCs (Fig. 5e, f ). Thus, we asked whether Tagln2−/− BMDCs
(See figure on next page.)Fig. 3 Tagln2−/− DCs showed impaired migration into the lymph node. a Expression of chemokine receptors and adhesion molecules in WT or Tagln2−/− DCs. The in vitro migration assay was performed using a Boyden chamber, and the number of migrating cells was counted by flow cytometry. b Time‑lapse imaging of WT or Tagln2−/− DCs on Fn in response to CCL19 (200 ng/mL). Representative morphology was categorized into three groups according to projection, spreading, and migration. Normal protrusions and irregular membrane morphology in WT or Tagln2−/− DCs are indicated as blue and red arrows, respectively. Scale bar, 10 μm. c Schematic diagram of the experimental setup for c and d. c, d The same number of WT (green) or Tagln2−/− DCs (red) was injected into the footpads of WT recipient mice, and the number of migratory cells in the draining popliteal LNs was analyzed by flow cytometry (c) and fixed cryosections (d). Anti‑B220 was used to distinguish the B cell zone. Scale bar, 100 μm. All data represent the mean of three experiments ± SEM. *P < 0.01
Page 8 of 23Kim et al. J Hematol Oncol (2021) 14:43
80
60
100
No.
of m
igra
ting
cells
001(NL
gniniardot
µm2 )
20
0
40
0-102 10 2 10 3
0
-102
102
103
104
0.25
0.87
Draining LN
WT - Green0-10
210
210
3
0
-102
102
103
104
KO
- O
rang
e
0 0
0
Non-draining
*
0.8
0.6
1.0
% o
f mig
ratin
g ce
llsNL
gniniardot 0.2
0
0.4
CCL3
# of
mig
ratin
g ce
lls (x
103 )
- +0
2.4
0.8
1.2
1.6
2.0
0.4
CCL5- +
CCL19- +
CCL21- +
SDF1α- +
12
1
2
TG2 WT-DC: Green TG2 KO-DC: Red BCZ - BlueWT KO
Popliteal lymph nodeMerged
WT KO
*
*
*
WT KO
Popliteal LN
Footpad injection
( WT DC, KO DC)
Cell and tissue analysis of LN at 24 h
0 104 1050
20
40
60
80
100
KOWT
Cel
l cou
nts
Iso.ctlCXCR4
0 104 105
CCR7
*
* *
2.8
0 104 1050
20
40
60
80
100
0 104 105
ICAM-1LFA-1Morphology
Projection
Spreading
Migration
-
-
-
+++++
+++++
+++++
+
+++
+
WTKO40
30
50
% o
f mor
phol
ogy
10
0
20
0 min 3 min 6 min 9 min
12 min 15 min
*
*
*
18 min 20 min
Tagln2
-/-D
C
Tagln2
+/+ D
C
Cel
l cou
nts
Fluorescent intensity
a
c
d
b
Page 9 of 23Kim et al. J Hematol Oncol (2021) 14:43
would be unable to optimally activate antigen-spe-cific T cells to expand in vivo. To this end, C57BL/6 mice were injected with OVA257–264-pulsed WT or Tagln2−/− BMDCs via the footpad. After 7 days, CD3+ T cells were purified and examined for proliferation and cytokine secretion in response to the same OVA257–264-pulsed WT BMDCs in vitro. As shown in Fig. 5g, h, Tagln2−/− BMDCs induced lower levels of cell pro-liferation and cytokine secretion, suggesting that they could not optimally support T cell clonal expansion in vivo.
Deletion of transgelin‑2 reduces DC contact with T cellsT cell activation and differentiation require sustained interaction with cognate DCs via integrin affinity and avidity regulation. In a previous study, we showed that transgelin-2 in T cells is associated with LFA-1 and can regulate LFA-1 avidity [17]. We therefore tested whether transgelin-2 is also involved in DC-mediated T cell adhe-sion. However, SEM analysis revealed no differences between WT and Tagln2−/− BMDCs (Fig. 6a). To assess the adhesion strength between BMDCs and T cells in a quantitative manner, we performed a conjugation assay in the presence of an OVA peptide. We found that Tagln2−/− BMDCs showed a significant reduction in con-jugation (Fig. 6b), demonstrating that transgelin-2 sup-ports adhesion between DCs and antigen-specific T cells.
To evaluate the role of transgelin-2 in regulating inter-actions between DCs and T cells in the live LN, we per-formed three-dimensional live-imaging of DCs using two-photon microscopy. OVA323–339-pulsed WT or Tagln2−/− BMDCs labeled with Cell Tracker CMFDA were s.c. injected into the rear footpads of C57BL/6 mice. At 24 h post-injection, OTII CD4+ T cells labeled with CMRA were i.v. injected (Fig. 6c). As previously observed (Fig. 3d), a significantly smaller number of Tagln2−/− BMDCs migrated into the LN as compared to WT BMDCs (Fig. 6c). Moreover, T cells formed more stable contacts with WT BMDCs than with Tagln2−/− BMDCs (Fig. 6d). As a result, the T cell speed in the LN was reduced, and track displacement differences were not significant (Fig. 6d). Collectively, these results demon-strate that transgelin-2 is essential not only for the deliv-ery of antigen materials through the dynamic movement of DCs but also for maintaining sustained interactions
between DCs and T cells for the initiation of adaptive immune responses.
Cell‑permeable recombinant transgelin‑2 fused with protein transduction domain (PTD) reconstitutes DC functionsIn a previous report, we demonstrated that cell-perme-able recombinant transgelin-2 fused with PTD (TG2P) enhanced cytotoxic T cell-mediated anticancer activ-ity through increased LFA-1 avidity [17], suggesting that TG2P can compensate for endogenous transgelin-2 without viral transduction. WT-TG2P was rapidly inter-nalized into the DCs (Fig. 7a). Figure 7b shows a sche-matic of the secondary structure of transgelin-2 fused with PTD. However, because we expected that natural transgelin-2 could be degraded by the ubiquitin pathway, as predicted by the program UbiSite [33], we substituted the potential ubiquitination site lysine (K)78 with argi-nine (R) to generate de-ubiquitinated TG2P (dU-TG2P) (Fig. 7b). Interestingly, dU-TG2P (K78R) showed remark-able stability and persisted longer than 24 h in the cyto-sol of BMDCs (Fig. 7c). Therefore, we next asked whether dU-TG2P could mimic the actions of transgelin-2 in Tagln2−/− BMDCs. To this end, Tagln2−/− BMDCs were incubated with dU-TG2P (10 μM) for 2 h, and then, the cells were seeded on Fn-coated plates. After 90 min, the size of the BMDCs was determined by flow cytometry, revealing that dU-TG2P significantly increased the size of Tagln2−/− BMDCs (Fig. 7d). To rule out a potential effect of LPS contamination and to confirm the specific-ity of the recombinant transgelin-2, dU-TG2P was heated inactivated (heat = H, H/dU-TG2P), and its effects on Tagln2−/− BMDCs were examined. Heat inactivation completely abolished the ability of dU-TG2P to increase the size of Tagln2−/− BMDCs (Fig. 7d), suggesting that the efficacy of dU-TG2P was solely mediated by transge-lin-2. dU-TG2P (10 μM) also significantly increased the spreading of Tagln2−/− BMDCs on Fn, whereas H/dU-TG2P had a little effect as determined by confo-cal microscopy and image analysis (Fig. 7e). Consist-ently, dU-TG2P remarkably increased T cell adhesion to Tagln2−/− BMDCs in a dose-dependent fashion (Fig. 7f ). Further, dU-TG2P-treated Tagln2−/− BMDCs supported more rapid antigen-specific (OT-II) T cell proliferation
Fig. 4 Tagln2−/− DCs do not optimally support T cell activation in vivo. a Schematic diagram of the experimental setup for this figure (left). WT or Tagln2−/− DCs were pulsed with pOVA (323–339), stained with CMFDA‑green, and injected into the footpad of OTII recipient mice. A representative photograph of excised popliteal LNs (right). Scale bar, 1 μm. b Flow cytometric plots show activation of migrating DCs in vivo at 24 h post‑injection. DCs were isolated from the popliteal LNs of recipient mice and stained with anti‑CD40 or CD80. The percent of CD11c+ cells in the draining LNs is presented as a bar graph. c DC‑mediated T cell activation in vivo. At 24 h post‑injection of DCs, OTII CD4+ T cells were isolated from the popliteal LNs of recipient mice and stained with anti‑CD69 or CD25. All data represent the mean of three experiments ± SEM. NS, not significant. *P < 0.01
(See figure on next page.)
Page 10 of 23Kim et al. J Hematol Oncol (2021) 14:43
- +
- +%
of C
D11
c+ cel
ls
CD40
0
1.0
0.2
0.4
0.6
0.8
pOVA 323-339
DC-injected DLN
WT Non-DL KO
4
3
5
Siz
e of
dr
aini
ng L
N (m
m)
1
0
2
WT KO
*
CD
69 M
FI
200
600
800
1000
400
0
WTKO
*
- +
Tagln2-/- DC Tagln2+/+ DC
0 104 1050
20
40
60
80
100
+ pOVAw/o pep
0 104 105
CD69 - PE
Cel
l cou
nts
42% 24%
0 104 1050
20
40
60
80
100
+ pOVAw/o pep
0 104 105
CD25 - PercpCy5
7.8%19.8%
CD
25 M
FI
100
200
250
300
150
0 - +OVA 323-339
50
*
Cel
l cou
nts
0 103 104 105
0-10
2
102
103
104
105
0 103 104 105
0-10
2
102
103
104
105
0.23 0.10
Tagln2-/- DC Tagln2+/+ DC
- +
% o
f pos
itive
cel
ls
0
50
10
20
30
40
CD80
*
*
0 103 104 105
0-10
2
102
103
104
105
0 103 104 105
0-10
2
102
103
104
105
CD
80
0.21 0.10
0.40 0.23
CMFDA-Green
CD
40
CMFDA-Green
NSNS
Popliteal LN Cell and tissue analysis of LN at 24 h
Footpad injection of pOVA323-339-pulsedWT or KO DC to OTII TCR mouse
WTKO
WTKO
a
b
c
Page 11 of 23Kim et al. J Hematol Oncol (2021) 14:43
and cytokine production than untreated or H/dU-TG2P-treated Tagln2−/− BMDCs (Fig. 7g, h).
dU‑TG2P potentiates DC‑based cancer immunotherapyAlthough we have previously shown that TG2P could enhance cytotoxic T cell-mediated anticancer activity [17], its efficacy may be limited unless a sufficient num-ber of tumor-specific T cell clones are obtained. From this point of view, the amplification of the DC func-tions may be more important because they can gener-ate numerous T cell clones targeting tumor antigens. We therefore asked whether dU-TG2P could promote the efficacy of WT BMDCs, which would prove use-ful for DC-based therapeutic applications. WT BMDCs treated with dU-TG2P exhibited significantly increased spreading on Fn (Fig. 8a). Moreover, dU-TG2P treatment produced increased adhesion between WT BMDCs and activated T cells (Fig. 8b), which stimulated the release of increased levels of T cell cytokines (Fig. 8c). Likewise, a larger number of T cells proliferated with co-cultured dU-TG2P-treated BMDCs than with untreated BMDCs (Fig. 8d).
We further asked whether dU-TG2P also could pro-mote the efficacy of WT cDC1s. Similar to the result of WT BMDCs, dU-TG2P increased conjugation between CD8+ OTI T cells and WT cDC1s (Additional file 1: Fig. S2A). In addition, increased conjugation correlated with the increased cytokine productions (Additional file 1: Fig. S2B). Accordingly, a larger number of T cells prolifer-ated with co-cultured dU-TG2P-treated cDC1s than with untreated cDC1s (Additional file 1: Fig. S2C).
To evaluate the effects of dU-TG2P-treated BMDCs in the in vivo tumor model, OVA257–264-pulsed untreated BMDCs or dU-TG2P-treated BMDCs were i.v. injected into mice, followed by OVA+B16F10 melanoma cells, as described in Fig. 1g. A significant reduction in metastatic nodules was observed in mice with adoptively trans-ferred OVA257–264-pulsed dU-TG2P-treated BMDCs compared to OVA257–264-pulsed untreated BMDCs (Fig. 8e). Consistent with the metastatic model, dU-TG2P-treated BMDCs significantly suppressed tumor growth in mice (Fig. 8f ). Kaplan–Meier survival studies
showed that mice injected with OVA257–264-pulsed BMDCs had a median survival time of 43 days and mice adoptively transferred with OVA257–264-pulsed dU-TG2P-treated BMDCs had an 80% probability of sur-viving longer than 47 days (Fig. 8g). Collectively, these results indicate that dU-TG2P is a promising therapeutic approach for DC-based cancer immunotherapy.
DiscussionAdaptive cellular immunity is initiated by the presenta-tion of a foreign antigen by DCs to antigen-specific naive T lymphocytes [22]. In the periphery upon pathogen encounter, immature DCs uptake antigen and proceed through a maturation process during their migration to the draining LNs. All aspects of immature and mature DC functions rely on dynamic rearrangements of the actin cytoskeleton, which are regulated by various actin-binding proteins and signaling pathways [34]. Despite the importance of DC migration from the periphery to the draining LNs, the roles of the numerous actin regula-tory molecules that control this process are incompletely understood. In this study, we showed that transgelin-2 is a critical actin-binding protein that supports the migra-tion of DCs to the draining LNs and DC-dependent priming of T cells for clonal proliferation, which are important functions for the host defense against foreign invaders and neoplastic diseases. Interestingly, recom-binant transgelin-2 protein, engineered for cell-penetra-tion and de-ubiquitination, significantly improved the therapeutic activity of WT BMDCs in controlling tumor growth and metastasis in mice.
We previously found that transgelin-2 expression increases in macrophages in response to LPS stimulation [19]. Among three transgelin family members, transge-lin-2 is the only isoform that contains an NF-κB con-sensus motif in the 5′ promoter region and is expressed in immune cells [19], suggesting that this small pro-tein plays a central role in host defenses against infec-tions and neoplastic diseases. We demonstrated that the actin–transgelin-2–LFA-1 axis in cytotoxic CD8+ T cells is effective in potentiating adoptive T cell therapy in cases where cancer cells express ICAM-1 on their surface
(See figure on next page.)Fig. 5 Tagln2−/− DCs did not optimally support T cell activation in vitro. a, c Differentiation (a), activation (b), and cytokine secretion (c) of Tagln2−/− BM cells. WT or Tagln2−/− BM cells were cultured with GM‑CSF, and then, CD11c+ cell populations were examined at the indicated days (a). Differentiated CD11c+ cells were further activated with LPS or pOVA (323–339), plus OTII T cells. Activation markers (b) and cytokine production (c) were determined. d–f The cells from a were co‑incubated with OTII CD4+ T cells in the presence of different doses of pOVA (323–339, 10−3–101 μg/mL), and then, T cell activation (CD69 and CD25 and IL‑2, IL‑4, and IFN‑γ) was determined by flow cytometry and ELISA. g Schematic diagram of the experimental setup for G and H. Representative histogram showing the in vitro proliferation of OVA (257–264)‑specific CD8+ T cells isolated from C57BL/6 mice administered with pOVA (257–264)‑pulsed WT DCs or Tagln2−/− DCs. Isolated CD3+ T cells were stained with CTV and co‑incubated with pOVA (257–264)‑pulsed WT DCs in vitro for 4 days. h From the supernatant of experiment g, IL‑2 or IFN‑γ production was determined at 24 h by ELISA. All data represent the mean of three experiments ± SEM. *P < 0.05; **P < 0.01
Page 12 of 23Kim et al. J Hematol Oncol (2021) 14:43
IL-2
(pg/
ml)
Conc. (µg/ml)
300
200
100
400
4000
3000
2000
5000
1000
8000
10000
6000
4000
2000
0
IFN
-γ (p
g/m
l) 40
30
20
10
50
+pOVA 0
800
600
400
1000
200
2500
3000
2000
1500
1000
500
WTKO
CD
69 M
FI
1500
250030003500
2000
1000500
CD
25 M
FI
1500
2000
2500
1000
500
*
100
IL-4
(pg/
ml)
20
80
60
40
120
pOVA+C
D4NT
pOVA+C
D4NT
pOVA+C
D4NT
10-3 10-2 10-1 100 101
WTKO
WTKO
Conc. (µg/ml)+pOVA 0 10-3 10-2 10-1 100 101
*
*
*
****
***
*
** ** **
T cell isolation for in vitroproliferation and activationtest on pOVA257-264-pulsed WT-DCs
MH
C c
lass
II M
FI
8000
160002000024000
12000
CD
80 M
FI
CD
86 M
FI
CD
40 M
FI
0
100
IL-1β
(pg/
ml)
20
806040
120100
IL-1
2 (p
g/m
l)
20
806040
120
40000
Time (h)
4000
80001000012000
6000
20000
Time (h)
8000
160002000012000
12000
40000
Time (h)
4000
80001000012000
6000
20000
Time (h)
- LPS
+ LPS
- LPS
+ LPS
- LPS
+ LPS
- LPS
+ LPS
LPS
WTKO
NS
10104
103
0 10104
103
05 5
81.2
72.1
48.5
21.6
6.15
CD11C - APC
Cel
l cou
nts
Day 1
Day 3
Day 5
Day 7
Day 9
WT KO
88.0
71.1
51.1
23.0
5.4
pOVA+C
D4NTLP
S
pOVA+C
D4NT
NS
NSNS
NS
NS
NS
NS
0
800
2400
3200
4000
1600
0
- pOVA257-264+ pOVA257-264
IL-2
(pg/
ml)
600
1800
2400
3000
1200
0
IFN
-γ (p
g/m
l)
Ctrl WT KO
**
**
0 102 103 104 105
stunoclleC
CTV
Ctrl
wt-DC
KO-DC
CD8+ T cell clonesPopliteal LN
i.v. injection (pOVA257-264-pulsed WT-DC or KO-DC)
WTKO
a
c
e
g h
f
d
b
Page 13 of 23Kim et al. J Hematol Oncol (2021) 14:43
14.3 4.54
79.41.770 103 104 105
0-10
2
102
103
104
105
15.8 7.29
75.31.57
21.7 7.33
69.41.76
21.6 10.5
66.31.61
0-10
2
102
103
104
105
0-10
2
102
103
104
105
0-10
2
102
103
104
105
0 103 104 105
0 103 104 105 0 103 104 105
1 h 2 h
DC
- O
rang
eT - Green
DCTDC
T
DC / CD4+ OTII T cells
Tagln2-/- DC
Tagln2+/+ DC
pOVA323-339-pulsed Tagln2-/- DC
pOVA323-339-pulsed Tagln2+/+ DC
24 h
OT II CD4 T cellsi.v. injection
Footpad injection of pOVA323-339-pulsed wt-DC or KO DCs to C57BL/6 mouse
OVA(323-339)-Tagln2+/+ DC / OT-II CD4+ T cells
0 min 11 min 22 min
OVA(323-339)-Tagln2-/- DC / OT-II CD4+ T cells
0 min 11 min 22 min
Tagln2-/- DC Tagln2+/+ DC
0
20
Time (h)
0 1
15
10
% o
f con
juga
tion
5
2
*
*
CCCCCC
*
WT KO
T ce
ll sp
eed
(µm
/s)
0.00
0.05
0.10
0.15
0.20
WT KO-2
0
2
4
6
Num
ber o
f adh
ered
CD
4+ T c
ells
/ D
C
1
3
5
-1
*
0
1000
2000
3000
4000
5000
Con
tact
dur
atio
n (s
ec)
WT KO
*
Num
ber o
f DC
s pe
r fie
ld
WT KO0
10
30
40
20
Num
ber o
f CD
4+ T c
ells
per f
ield
WT KO0
10
20
40
50
30
WTKO
**
b
d
a
c
Fig. 6 Tagln2−/− DCs do not fully support T cell adhesion in vitro and in vivo. a SEM image of a representative DC‑T cell interaction. WT or Tagln2−/− DCs were pulsed with pOVA (323–339) and co‑incubated with OTII CD4+ T cells for 1 h. Scale bar, 5 μm. b Reduced conjugate formation in Tagln2−/− DCs. Stable conjugates were identified as double‑positive events by flow cytometry. c Schematic diagram of the experimental setup for c and d. Representative cryosection images showing the overall distribution of WT or Tagln2−/− DCs (green) and OTII CD4+ T cells (red) in draining lymph nodes. The statistical analysis of the number of migrated CD4+ T cells or DCs was performed. Scale bar, 100 μm. d Representative snapshot of live images of DC–T cell interactions in vivo. Draining popliteal LNs were visualized by two‑photon microscopy. Scale bar, 10 μm. White arrowheads indicate the contact cells. Statistical analysis of the number of T cells in contact with one DC, the contact duration, and the T cell speed are presented. All data from b–d represent the mean of three experiments ± SEM. *P < 0.05
Page 14 of 23Kim et al. J Hematol Oncol (2021) 14:43
[17]. Indeed, LFA-1 is an essential initiator for the for-mation of the IS between cytotoxic T cells and cancer cells, and it mediates the polarization of cytotoxic gran-ules toward target cells via tight adhesion to the target cells [35]. However, not all cancer cells express ICAM-1 [17]. Moreover, some reports have demonstrated that the expression of ICAM-1 is positively correlated with a more aggressive tumor phenotype and metastatic poten-tial [36, 37]. By contrast, the antitumor functions of DCs are mediated through the initiation of various adaptive immune mechanisms, including clonal expansion of antigen-specific CD4 and CD8 T cells. Thus, improving DC functions represents a more attractive strategy than directly enhancing T cell functions. In this respect, cell-permeable peptides that promote transgelin-2-like func-tions in DCs have a potential clinical value as a cancer immunotherapy based on DCs.
In some cancer cells, transgelin-2 is known to inhibit cellular motility by suppressing actin polymerization [38]. Consistently, transgelin-2 was found to be more downregulated in metastatic tumors than in primary can-cers [38]. However, as observed in this study, the reduced migration of Tagln2−/− BMDCs toward chemokine gra-dients or into the draining LN unambiguously suggests that transgelin-2 is involved in the dynamic movement of DCs. This conclusion is also corroborated by our previ-ous works, in which transgelin-2- KO in T cells or mac-rophages reduced their motility [16, 19]. Dynamic actin regulation by transgelin-2 appears to be mediated by its ability to induce small filopodia-like protrusions at the leading edge of migrating cells and to control podosome formation [27]. Filopodia and podosomes are impor-tant subcellular architectures that sense the external environment and degrade the ECM during DC migra-tion, respectively [27]. Moreover, the fact that Tagln2−/− BMDCs showed a remarkable decrease in F-actin levels suggests that transgelin-2 is involved in actin polymeri-zation in vivo [16]. We believe that the reduced F-actin content in transgelin-2 KO cells is due to the rapid decomposition of polymerized F-actin as this protein directly stabilizes F-actin structures after polymerization but does not increase actin polymerization [16, 18, 39].
In cancer cells, however, these characteristics of transge-lin-2 may be involved in the process of tumorization in a wide range of cancers [14, 40]. In this respect, transge-lin-2 may be a promising target protein for cancer ther-apy. In fact, several reports using chemical compounds or microRNAs targeting the Tagln2 gene have shown poten-tial positive results in the suppression of cancer devel-opment and metastasis. These interesting features of transgelin-2 suggest that this small actin-binding protein acts as a double-edged sword in the context of cancer and immune cells.
One interesting lingering question is the mechanism by which transgelin-2 in BMDCs mediates increased T cell adhesion, thereby enhancing T cell clonal proliferation. One possibility is that transgelin-2 may participate in the growth of small microvilli on the DC surface, and these multiple finger-shaped structures could provide a physi-cal means of clustering adhesion molecules to support T cell adhesion. Interestingly, a previous report demon-strated that DCs can produce multifocal synapses with clustered T cells via microvilli [41]. These microvilli on the DCs exhibited a high density of antigen-presenting molecules and co-stimulatory molecules, providing the physical basis for the preferential adhesion of both CD4+ and CD8+ T cells [41, 42]. Along these lines, Jung et al. and our group recently found that T cell microvilli also provide a platform to cluster important T cell molecules, including TCR, TCR complex, co-receptors, and co-stimulatory molecules [43, 44], suggesting that initial rec-ognition and adhesion are mediated through polarized microvilli between DCs and T cells.
DCs are the most potent antigen-presenting cell type and are key players in tumor-specific immune responses. This characteristic has been exploited by DC therapy, in which DCs are loaded with tumor-associated antigens and applied to patients to induce immune responses against tumor antigens. However, although multiple clinical trials have been performed, clinical scores have been largely disappointing. This is due in part to insuf-ficient antigen presentation and T cell activation, migra-tory potential, and cytokine production [45]. In this regard, accumulating evidence suggests cDC1s—which
(See figure on next page.)Fig. 7 Recombinant dU‑TG2P reconstitutes Tagln2−/− DC functions. a The transduction efficiency of WT‑TG2P (10 µM) in WT or Tagln2−/− DCs. b Schematic diagram of the domain composition of transgelin‑2. A potential ubiquitination site, K78, is highlighted in red. c Stability test of WT‑TG2P and dU‑TG2P (K78R) in DCs. d, e Reconstitution of transgelin‑2 by dU‑TG2P. Tagln2−/− DCs treated with dU‑TG2P or heat‑inactivated dU‑TG2P (H/dU‑TG2P) were seeded on Fn‑coated plate, and the cell size (d) and spreading (e) were determined by flow cytometry and confocal microscopy, respectively. F‑actin was stained by phalloidin‑TRITC (E). Cell spreading areas were calculated using ImageJ software. f–h dU‑TG2P rescues transgelin‑2 function. Tagln2−/− DCs treated with H/dU‑TG2P (10 µM) or dU‑TG2P (5–10 µM) were co‑incubated with OTII CD4+ T cells in the presence or absence of pOVA (323–339), and then, the cells or cultured supernatants were subjected to a conjugates assay (F), a cytokine production assessment (g), and a proliferation test (h). All data from e–h represent the mean of three experiments ± SEM. NS, not significant. *P < 0.01
Page 15 of 23Kim et al. J Hematol Oncol (2021) 14:43
Ubi. motif
CH AB CR25
1
153 164199174136
TAGLNs
72EGQAPVKKIQASSM85 K78R
120Time (h)
dU-T
G2P
(K78
R)
β-actin
wt-T
G2P
4 18 24
α−His(TG2P)
wt-TG2P - + +2
+ ++
- + - +TG2
β-actin
Tagln2-/- DC Tagln2+/+ DC
45
22
PTD
PTD seq: QIL+GGG+YGRKKRRQRRR (C-term+linker+PTD)
120Time (h) 4 18 24dU-TG2P - + +
2+ ++
β-actin
0
30
20
10
- pOVA323-339+ pOVA323-339
% o
f con
juga
tion
45
22
45
22
M
M
M
Tagln2-/-Tagln2+/+
*
NS10
10
10
2
3
4
0
42.6 3.60
52.80
42.9 5.67
50.50.46
36.8 6.37
55.20.5242.5 9.01
48.40.12
42.6 15.5
41.90.040 10
210
310
4
Tagln2-/- DC
dU-TG2P (5)
-933-323
AV
Op+
933-323A
VOp
dU-TG2P (10)
)A
RM
C(C
D
T (CTV)
NT
0 102
103
104
38.9 6.68
54.30.070 10
210
310
4
102
103
104
0
32.3 6.77
54.40.01
46.2 3.99
48.60
0 102
103
104
FN (9
0 m
in)
Spr
eadi
ng a
rea
(µm
2 )
dU-TG2PNT H/dU-TG2P
H/dU-TG2P (10)
H/dU-T
10dU
-T5
dU-T
10NTNT
*
Tagln2-/- DC
0
100
200
300
400
500
600
Tagln2-/- DC (90 min)
NT
H/dU-T
G2P
dU-T
G2P
NS
P < 0.0001
P = 0.0023
0 50K 100K 150K 200KFSC
50
100
150
Cel
l cou
nt
NT H/dU-TG2P dU-TG2P
0 50K 100K 150K 200K 250KFSC
DIC/actin
0
500
1000
1500
NT0
500
1000
1500
IL-2
(pg/
ml)
IFN
-γ (p
g/m
l)
H/dU-T
10NTdU
-T5
dU-T
10
- OVA + OVAH/dU-TG2P (10)
102
103
104
0
dU-TG2P (5) dU-TG2P (10)
10
10
10
2
3
4
0
NT
53.937.9
25.520.5
Ki6
7+ cel
ls (%
)
0
20
40
60
80Tagln2-/- DC
Tagln2-/-
Tagln2+/+ H/dU
-T10dU
-T5
dU-T
10NT+ O
VA- O
VA
+ OVA
*
*
NS
Ki-6
7
NucSpotTagln2-/-
Tagln2
+/+
**
NS
*
*
NS
1 K 2 K 3 K 4 K0 1 K 2 K 3 K 4 K0
wt-TG2P
α−His(TG2P)
a
c
f
g h
d
e
b
Page 16 of 23Kim et al. J Hematol Oncol (2021) 14:43
are different from monocyte-derived DCs—play an inte-gral role in tumor immunity and are a good candidate for vaccination purposes [45]. In the present study, we found that transgelin-2 is also induced in Flt3 ligand-induced cDC1s. Moreover, reduction of tumor growth control by Tagln2−/− cDC1s strongly suggests that transgelin-2 is an important actin regulator for optimal action of vari-ous DC subsets. Therefore, it will be very interesting to investigate the global gene signatures of WT BMDCs and Tagln2−/− BMDCs and to compare these with cDC1s. Further, it will be interesting to test whether cell-per-meable transgelin-2 can change the gene signatures of BMDCs toward the cDC1s.
Abnormal changes in the actin cytoskeleton contrib-ute to the growth, metastasis, and invasion of cancer cells. However, because the actin cytoskeleton is indis-pensable for all living cells, drugs that target the actin cytoskeleton of tumor cells may exhibit off-target toxic-ity in noncancerous cells. To overcome this matter, tar-geting actin-regulating factors with altered expression in cancers may become an alternate therapy to increase tumor toxicity. Interestingly, transgelin-2 is essential for both cancer development and immune functions [23, 40]. This suggests that transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Transgelin-2 plays an important role in fine-tun-ing the structure and function of the actin cytoskeleton, which is crucial for DC migration, antigen presenta-tion, and the formation of the immune synapse between DCs and T cells. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy.
ConclusionAdaptive cellular immunity is initiated by a series of actions of DCs that uptake and present foreign antigens, migrate to the draining LNs, and interact with antigen-recognizing T cells. Transgelin-2, a 22 kDa actin-bind-ing protein, is upregulated in DCs during maturation and LPS activation. Tagln2−/− DCs exhibited significant defects in their abilities to home to draining LNs and to form optimal contacts with cognate CD4+ T cells to prime T cells, and these changes were associated with
a failure to suppress tumor growth and metastasis of B16F10 melanoma cells in mice. Recombinant transge-lin-2 protein, engineered for cell-penetration and de-ubiquitination, potentiated DC functions to suppress tumor growth and metastasis, demonstrating that this small-actin binding protein represents a promising thera-peutic approach for DC-based cancer immunotherapy.
Materials and methodsAntibodies and reagentsRabbit polyclonal anti-transgelin-2 antibody was raised in rabbits using purified full-length transgelin-2 (AbFron-tier, Seoul, Korea). In addition, the following antibodies were used: goat polyclonal anti-TAGLN1 (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-β-actin; rabbit polyclonal antibodies against p-PI3K, t-PI3K, p-AKT, t-AKT, p-p38, t-p38, p-ERK, t-ERK, His, HRP-conjugated anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-TAGLN3 and anti-vinculin (Abcam, Cambridge, MA, USA); and antibod-ies for FITC-conjugated CD40 (MA5-16506), MHCII (11-5322-82), CD18 (LFA-1β; 11-0181-82), ICAM-1 (11-0541-82), CD11c (17-0114-82), PE-conjugated CD86 (12-0862-82), CD80 (12-0801-82), CD25 (120251-82), CD69 (12-0691-82), CXCR4 (12-9991-82), CCR7 (12-1971-82), APC-conjugated CD11c (17-0114-82), and B220 (17-0452-82) (eBioscience, San Diego, CA, USA); FITC-con-jugated CD24 (M1/69), CD103 (2E7), and SIRPα (P84) (BioLegend, San Diego, CA, USA). All antibodies for flow cytometry were used at a dilution of 1:100. Phalloi-din-TRITC, lipopolysaccharide, and Fn were purchased from Sigma Aldrich (St. Louis, MO, USA). GM-CSF, CCL-3, CCL-5, CCL-19, CCL-21, and SDF-1α were pur-chased from Peprotech Inc. (Rocky Hill, NJ). Alexa647-phalloidin, CellTracker CMFDA-green, CMRA-Orange dyes, anti-mouse Alexa 647, and anti-rabbit Alexa488 were purchased from Invitrogen (Carlsbad, CA, USA). A CellTrace™ Violet (CTV) Cell Proliferation Kit was pur-chased from Thermo Fisher Scientific (Waltham, MA, USA). OVA peptide fragments (323–339 and 257–264) were purchased from GeneScript (San Francisco, CA, USA). Flt3L-Ig was purchased from Bio-X-Cell (West
Fig. 8 Recombinant dU‑TG2P potentiated DC‑mediated tumor therapy. a dU‑TG2P enhances DC cell spreading on Fn. WT DCs treated with dU‑TG2P (10 µM) were placed on Fn‑coated plates, and the spreading area was measured using ImageJ. Scale bar, 10 μm. b–d OTII CD4+ T cells were co‑incubated with nothing or with dU‑TG2P‑treated WT DCs pulsed with pOVA (323–339), and then, the cells or cultured supernatants were subjected to a conjugate assay (b), cytokine production assessment (c), and proliferation test (d). e, f Gross images of OVA+B16F10 lung and solid tumors, respectively. C57BL/6 mice were injected i.v. with media alone, DCs, or dU‑TG2P‑treated DCs pulsed with pOVA (257–264). After 7 days, the mice were i.v. (e) or s.c. (f) injected with OVA+B16F10 cells. The metastatic nodules (e) and tumor sizes and weights (f) were measured after 8 days of OVA+B16F10 injection. g The survival rates of tumor‑bearing mice post‑implantation are shown. All data represent the mean of three experiments ± SEM. *P < 0.01
(See figure on next page.)
Page 17 of 23Kim et al. J Hematol Oncol (2021) 14:43
CD
+C
D -Ud
P2GT
60 120 150 180900 30
*
No. of nodules
DC + dU-TG2P
DCMOCK
0
0.1
0.2
0.3
0.4
0.5
0.6
0
0.4
0.8
1.2
1.6
Tum
or w
eigh
t (g)
Tum
or s
ize
(cm
)33.0 3.15
63.00.93
102
103
104
0
29.4 13.5
56.70.3533.7 5.50
60.30.46
102
103
104
0
32.9 20.4
45.71.030 10
2 103 104
CD
)A
RM
C(
T (CTV)0 102 103 104
enoN
P2GT-
Ud
- pOVA323-339 + pOVA323-339
0
500
1000
1500
2000
2500
0
500
1000
1500
2000
None
dU-T
G2P
dU-T
G2P
dU-T
G2P
dU-T
G2P
* *
IL-2
(pg/
ml)
IFN
-γ (p
g/m
l)
+ OVA + OVA
58.3
dU-TG2P (2 days)
1 K 2 K 3 K 4 K0
32.5
None (2 days)
0102
103
104
1 K 2 K 3 K 4 K0
Ki6
7+ cel
ls (%
)
0
20
40
60
80
- OVA
None
*
dU-T
G2P
Ki-6
7
NucSpot
101
102
103
104
105
Cel
l cou
nt
T (CTV)
None (5 days)dU-TG2P (5 days)
MOCK DC
MOCK DC
Sur
viva
lpro
babi
lity
(%)
00
25
50
75
100
Days
DCDC+dU-TG2P
30 35 40 45 50
P =
0.00
31
P <
0.00
01
DC
+ dU-T
G2P
wt-DC
wt-DC + dU-TG2P
None
None
None
None
None *
dU-TG2P
dU-TG2P0 8 16 24 32 40
% of conjugation
+ OVA
Spr
eadi
ng a
rea
(µm
2 )
0
300
600
900
1200
1500
dU-T
G2PNon
e
90 minP < 0.0001
0 min 90 min
0 min 90 min
Phase contrasts
enoN
P2GT-
Ud
- OVA
+ OVA
DC
+ dU-T
G2P
a
d
f
e
g
c
b
Page 18 of 23Kim et al. J Hematol Oncol (2021) 14:43
Lebanon, NH, USA). CD45R (B220) MicroBeads was purchased from Miltenyi Biotec. (Bergisch Gladbach, Germany).
CellsB16F10 (CRL-6475) cell lines were purchased from ATCC. A stable B16F10 cell line expressing membrane-bound OVA (OVA+B16F10) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cam-bridge, MA) using Lipofectamine 2000 reagent (Invit-rogen) and selection with G418 (InvivoGen, San Diego, CA, USA). For BMDCs cultures, 5 × 106 BM cells were cultured in 10 mL of RPMI supplemented with 20 ng/mL recombinant murine GM-CSF for 7 to 9 days. GM-CSF was added every 3 days. To generate cDC1s, 3 × 106 BM cells were incubated in 3 mL of RPMI supplemented with 200 ng/ mL Flt3-L for 9 days. Flt3-L was added every 2 days and cDC1s (CD11c+B220−) were isolated by anti-B220 positive selection beads to exclude plasmacytoid DCs (CD11C+B220+) for further experiments. However, unless otherwise indicated (for Additional file 1: Figs. S1 and 2), we used GM-CSF-induced BMDCs for most of the experiments. Naive CD4+ T cells were purified from the mouse spleen and LNs by negative selection using an EasySep magnetic separation system (Stemcell Tech-nologies, Vancouver, Canada). To generate mouse T cell blasts, OTII CD4+ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured further for 3 days with 100 U/mL rIL-2.
MiceC57BL/6 wi mice and OTII TCR transgenic mice (C57BL/6 background) were purchased from Damul Sci-ence (Korea) and Jackson Laboratories (Bar Harbor, ME, USA), respectively. All mice were housed under specific pathogen-free conditions. Transgelin-2 (Tagln2−/−) KO mice have been described previously [16]. All experimen-tal methods and protocols were approved by the Institu-tional Animal Care and Use Committee of the School of Life Sciences, Gwangju Institute of Science and Technol-ogy, and carried out in accordance with their approved guidelines (IACUC GIST-2015–04).
Western blottingTo analyze transgelin family expression in DCs, BM cells were harvested at the indicated day during differentia-tion with GM-CSF and Flt3L, respectively, and cells were lysed in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, and one tab-let of complete protease inhibitors) for 15 min on ice. Cell lysates were centrifuged at 16,000 × g for 30 min at 4 °C, and the supernatants were eluted with sodium dodecyl
sulfate (SDS) sample buffer (100 mM Tris–HCl, pH 6.8, 4% SDS, and 20% glycerol with bromophenol blue) and heated for 5 min. The proteins were separated by SDS polyacrylamide gel electrophoresis on 10%–15% gels and were transferred to nitrocellulose membranes using a Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA). The membrane was blocked in 5% skim milk for 1 h, rinsed, and incubated with the appropriate antibod-ies in TBS containing 0.1% Tween 20 (TBST) and 0.5% skim milk overnight. Excess primary antibody was then removed by washing the membrane three times in TBST. The membrane was then incubated with 0.1 μg/mL per-oxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse) for 1 h. After three washes with TBST, bands were visualized using western blotting detection reagents (EZ-Western Lumi Femto Kit; DoGenBio, Seoul, South Korea) and were then exposed to an X-ray film (Kodak, Rochester, NY).
Analysis of differentiation and activation of DCsWT or Tagln2−/− BMDCs (1 × 106) were activated with 200 ng/mL of lipopolysaccharide (LPS), harvested, and blocked with a rat anti-mouse CD16/CD32 antibody (mouse Fc Block, BD Pharmingen). Cells were then stained with activation markers, including CD11c, CD80, CD86, CD40, and MHC-II, for flow cytometry. To exam-ine T cell-mediated DC activation in vitro, 1 μg/mL of pOVA (323–339)-pulsed WT or Tagln2−/− BMDCs (1 × 105) was co-cultured with OTII CD4+ T cells (5 × 105) for 24 h, and the supernatants were subjected to ELISA assay to examine cytokine production.
Cell spreading on FnBMDCs were plated on coverslips coated with or with-out 10 μg/mL of Fn for 90 min. The cells were fixed for 10 min with 4% paraformaldehyde and permeabilized with 0.1% Triton-X (Sigma-Aldrich) in PBS for 10 min at room temperature (RT). The coverslips were then incubated with TRITC-conjugated phalloidin at RT for 30 min, washed, mounted onto slide glass using Vectash-ield (VectorLabs, Burlingame, CA), and imaged using a FV-1000 confocal microscope (Olympus, Tokyo, Japan) To measure cell spreading area, the captured images were analyzed using ImageJ software (NIH) as follows: threshold values were set to define the cell edge, and a mask was then created for each cell to get the total cell area (with arbitrary units) within the mask. Cell size was determined by flow cytometry after detachment of the BMDCs with 10% EDTA.
Conjugation assayOTII CD4+ or OTI CD8+ T cells were stained with Cell Tracker Green CMFDA, and WT or Tagln2−/− BMDCs
Page 19 of 23Kim et al. J Hematol Oncol (2021) 14:43
or Flt3L-induced cDC1s were stained with Cell Tracker Orange CMRA for 30 min. The cells were then washed and resuspended in RPMI 1640 media. For conjuga-tion, DCs were incubated with T cells (1:5 ratio) for 2 h in the presence or absence of pOVA (323–339, 1 μg/mL) or pOVA (257–264, 1 μg/mL). The relative proportion of green, orange, and green and orange-positive events in each tube was determined by FACS Canto (BD Bio-sciences, San Jose, CA) and analyzed with FlowJo soft-ware (Treestar, San Carlos, CA). The number of gated events counted per sample was at least 10,000. The per-centage of conjugated T cells was determined as the number of dual-labeled (green and orange-positive) events divided by the number of green-positive T cells.
Determination of in vitro and in vivo T‑cell activationTo examine in vitro T-cell activation, the indicated con-centration of pOVA (323–339, 10−3–101 μg/mL)-pulsed WT or Tagln2−/− BMDCs (1 × 105) was co-cultured with OTII CD4+ T cells (5 × 105) for 24 h, and the super-natants were subjected to ELISA assay to determine cytokine secretion. In addition, the cells were stained with anti-CD69 or CD25 to determine DC activation. To determine the effects of dU-TG2P, BMDCs or Flt3L-induced cDC1s (1 × 105) were treated with dU-TG2P along with pOVA (323–339, 1 μg/mL) or pOVA (257–265, 1 μg/mL) for 2 h and co-cultured with OTII CD4+ T cells (5 × 105) or OTI CD8+ T cells (5 × 105) for 24 h, and the supernatants were subjected to ELISA assay to deter-mine cytokine secretion. For in vivo T-cell priming, WT or Tagln2−/− BMDCs (3 × 106) were pulsed with pOVA (323–339, 1 μg/mL) for 2 h and injected s.c. into the footpads of OTII mice, and the cells were isolated from the popliteal lymph node at 24 h post-injection. In gen-eral, to stain the cells with fluorescently conjugated anti-bodies, the cells were blocked with anti-FcγR antibody (CD16/32, clone 2.4G2) and then stained for surface acti-vation markers. Data were acquired using a FACS Canto and analyzed with FlowJo software.
In vitro migration assay using a Transwell systemTranswell cell migration was assayed using a 96-well Boyden chamber (ChemoTx plate, Neuroprobe, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. The Boyden chamber was assembled with polyvinylpyrrolidone-free polycarbonate filters (3–5-µm pore size). WT or Tagln2−/− BMDCs (1 × 106) were added to the Fn-coated upper compartment, and media containing 200 ng/mL of each chemokine were added to the lower compartment. The apparatus was incubated for 4 h at 37 °C in a humidified CO2 incubator. Cells on the bottom wells were harvested and resuspended in 300 µL of PBS, and the number of cells was counted using a
FACS Canto for a fixed period of time (300 s) under con-stant middle pressure.
In vivo migration assayTo evaluate DC migration, a mixture of 2 × 106 WT (CMFDA-green) and 2 × 106 Tagln2−/− BMDCs (CMRA-orange) was injected into the footpads of WT recipi-ent mice. A popliteal lymph node was harvested at 24 h post-injection. The popliteal LNs were fixed in 4% para-formaldehyde in PBS at 4 °C overnight. On the next day, the samples were washed and incubated in PBS with 30% sucrose (w/v) (Sigma-Aldrich) overnight at 4 °C. The samples were then embedded in Tissue-Tek® O.C.T. Compound (Thermo Fisher Scientific) and frozen using 2-methylbutane, cooled with liquid nitrogen. 10 μm sec-tions were cut using a Leica CM1800 cryostat. For immu-nostaining, tissue sections were blocked for 2 h at RT in 10% normal goat serum (Sigma-Aldrich). The sections were incubated with fluorescently conjugated anti-B220 antibody at RT for 30 min in 10% goat normal serum. The samples were washed three times to remove unbound antibody and mounted in Permount solution (Thermo Fisher Scientific). Images were acquired with a confo-cal microscope and analyzed with Fluoview software. To quantify the number of migratory DCs, single-cell sus-pensions from the draining popliteal LNs were obtained by digestion in collagenase D, and the % of migrating DCs was quantified using FACS Canto. In some experiments, the excised lymph node was photographed for size deter-mination. Draining popliteal LNs were harvested from the left hind limb, which were injected with cells through the footpad, whereas nondraining LNs were excised from the right hind limb.
ImmunocytochemistryTo analyze the podosome dynamics, BMDCs were har-vested and seeded on poly-L-lysine-coated glass cover-slips in a 12-well plate (2 × 106 cells/well in supplemented medium) and incubated for the indicated time in the presence of 200 ng/mL of LPS at 37 °C and 5% CO2. To analyze the localization of transgelin-2, BMDCs were incubated for 24 h in the same condition. Coverslips were washed once with warmed PBS, followed by fixa-tion with 4% paraformaldehyde in PBS at RT for 10 min. After permeabilization using 0.1% Triton X-100, the cells were stained with 10 µg of anti-vinculin and anti-transge-lin-2 antibodies at 4 °C overnight. The next day, the cov-erslips were washed with PBS two times, and the cells were stained with mouse anti-Alexa647 and rabbit anti-rabbit Alexa488 secondary antibodies. For actin stain-ing, permeabilized cells were incubated with anti-Alexa 647- or TRITC-conjugated phalloidin (1:100) for 1 h at RT. The coverslips were mounted onto slideglass using
Page 20 of 23Kim et al. J Hematol Oncol (2021) 14:43
Vectashield (Vector Labs) and imaged using a confocal microscope. For quantitation of cells with podosomes, the cells were assessed for the presence of at least one clearly identifiable podosome with an F-actin-rich core. At least 100 cells were scored per sample, with a mini-mum of three biological replicates.
DC–T cell interactions via intravital two‑photon microscopyFor in vivo imaging, pOVA (323–339, 1 µg/mL)-pulsed WT or Tagln2−/− BMDCs (3 × 106) were stained with Cell Tracker CMFDA-green and injected s.c. into the footpads of WT recipient mice. After 24 h, purified OTII CD4+ T cells were stained with CMRA-orange and adoptively transferred to recipient mice intrave-nously. Mice were anesthetized with isoflurane, and the popliteal LNs were surgically exposed. Imaging was per-formed on a Zeiss LSM 880 microscope equipped with a MaiTai laser (Coherent) tuned to 750 nm in combination with an NDD2 BIG2 GaAsP detector and a 20 × water-dipping lens (NA 1.0, Zeiss) using ZEN v2.1 acquisition software. Images were collected with a typical voxel size of 0.593 × 0.593 × 1.0 μm and a volume dimension of 607.28 × 607.28 × 200 μm to create a three-dimensional data set. For four-dimensional data sets, images were col-lected with a typical voxel size of 0.45 × 0.45 × 1.5 μm and a volume dimension of 425.1 × 425.1 × 30 μm. This volume collection was repeated every 60 s for up to 2 h. To analyze the number of DC-contacted T cells and the speed, displacement, and duration of these interactions, tracks were generated for T cells and analyzed using Ima-ris software. Data were plotted using Prism (GraphPad).
Time‑lapse video microscopyFor the dynamic analysis of DC spreading and protru-sion, time-lapse imaging was conducted on an EVOS system (EVOS™ FL Digital Inverted Fluorescence Micro-scope, Fisher Scientific, Paisley, Scotland, UK). WT or Tagln2−/− BMDCs (3 × 105) were seeded on 10 µg/mL of Fn-coated 12-well non-tissue culture plates for 10 min at 37 °C, and the plates were immediately placed in the chamber of the EVOS unit (which was programmed to supply 5% CO2 and maintained at 37 °C constant temper-ature). The cells were recorded in the presence or absence of 200 ng/mL CCL19 every 10 s for up to 1 h. Sequential images were analyzed using ImageJ software.
Scanning electron microscopyFor scanning EM, cells were fixed with 2.5% glutaral-dehyde solution for 2 h, rinsed with PBS for 5 min, and fixed in OsO4 for 2 h. The samples were then dehy-drated through incubation with a graded ethanol series over 30 min and dried in a critical point dryer. The samples were prepared by sputter coating with 1–2 nm
gold–palladium and analyzed using FE-SEM (HITACHI, Tokyo, Japan).
B16F10 melanoma tumor model and isolation of TILsTo evaluate transgelin-2 functions in T cells for tumor suppression (for Fig. 1b), PBS (none), WT (OTI-T), KO (Tagln2−/− OTI-T), or transgelin-2-overexpressing T (TG2OE OTI-T) cells were adoptively transferred into tumor-bearing C57BL/6 mice at days 7, 10, and 13 post-tumor implantation. All mice were sacrificed at day 25, and tumors were isolated and weighed.
To evaluate the effect of Tagln2−/− in mice (for Fig. 1c), OVA+B16F10 cells (3 × 105) were s.c. injected into the dorsal flank region of age- and sex-matched WT or Tagln2−/− mice. To evaluate transgelin-2 functions in DCs for tumor suppression, BMDCs (1 × 107) from WT or Tagln2−/− were pulsed with 1 µg/mL of pOVA for 2 h and i.v. injected into 8-week-old WT mice. At day 7, OVA+B16F10 cells (3 × 105) were s.c. or i.v. injected into the dorsal flank region to induce solid and metastatic tumor models, respectively. Mice were sacrificed at day 8 post-inoculation with tumor cells. At the end of the experiments, tumors were isolated, weighed, and photo-graphed for gross morphology. To analyze TILs, tumor tissues were dissected and mechanically disaggregated before digestion with collagenase D (1 mg/mL, Roche) for 30 min at 37 °C. After digestion, all of the cells were passed through 70-µm filters, and leukocytes were iso-lated by centrifugation using 38% Percoll for 30 min. Pel-lets were resuspended with PBS, stained with anti-CD3 and CD8 antibodies, and analyzed by flow cytometry. To analyze cytokine production, isolated TILs were stimu-lated with PMA/Ionomycin (200 nM/ 1 µM) in the pres-ence of Brefeldin A (1 µg/mL) for 4 h at 37 °C. Cells were subsequently collected and stained for CD8 followed by fixation with IC fixation Buffer (eBioscience) for 20 to 30 min at RT. Then, the cells were washed twice with 1 × Permeabilization Buffer (eBioscience) and stained with IFN-γ antibody. After washing, cells were analyzed by flow cytometry.
Generation of OVA‑specific T cells and ex vivo proliferation assayTo measure proliferation and cytokine production from OVA-specific T cells, DCs from WT or Tagln2−/− (1 × 107) were pulsed with 1 µg/mL of pOVA (257–265) for 2 h and i.v. injected into 8-week-old C57BL/6 WT mice. At day 7, CD3+ T cells were isolated from the LNs and spleens, stained with CTV, and co-cultured with 1 µg/mL of pOVA (257–265)-pulsed WT BMDCs for 4 days for clonal expansion. The proliferative CTV-pos-itive cells that were in a live cell gate were quantified by flow cytometry. To measure the IL-2 or IFN-γ cytokines
Page 21 of 23Kim et al. J Hematol Oncol (2021) 14:43
from OVA-specific T cells, co-cultured supernatants were harvested at 24 h and subjected to ELISA.
Generation of dU‑TG2P mutant and purification of recombinant TG2P or dU‑TG2PRecombinant transgelin-2 protein fused with PTD (TG2P) was described previously [17]. To generate a de-ubiquitinated mutant of TG2P, a potential ubiquitinated amino acid residue was predicted using the program UbiSite [33], and the resulting residue, K78, was mutated to an R by site-directed mutagenesis. The resulting PCR-amplified cDNAs encoding dU-TG2P were ligated into the pET-28a vector (Novagen, Madison, WI). Expression of recombinant TG2P and dU-TG2P in Escherichia coli BL21 (DE3) cells has been described previously [17].
Introduction of TG2P (dU‑TG2P) into DCsDCs were washed and incubated with 10 μM of TG2P or dU-TG2P for 2 h at 37 °C in serum-free media. The cells were then washed with PBS and resuspended in media for further assays. To rule out the potential for LPS con-tamination, dU-TG2P was heat-inactivated by boiling for 5 min.
Proliferation assayOTII CD4+ or OTI CD8+ T cells were stained with CTV and co-cultured with 1 µg/mL of pOVA (323–339)-pulsed BMDCs or pOVA (257–264)-pulsed cDC1s (1 × 105) for 2, 3 or 5 days. Total cells were fixed with Cell Fixation & Cell Permeabilization Kit (Thermo Fisher Scientific) for 1 h at 4 °C and stained with anti-Ki67 along with 1 μL of NucSpot Far-Red for 1 h at 4 °C in the dark. The samples were washed with PBS and analyzed by flow cytometry. The percent of proliferative populations was acquired from the gate in a CTV-positive population.
StatisticsStudent’s t-test and one-way ANOVA analysis of variance (corrected for all pairwise comparisons) were performed using Prism software. P-values < 0.05 were considered statistically significant.
Supplementary InformationThe online version contains supplementary material available at https ://doi.org/10.1186/s1304 5‑021‑01058 ‑6.
Additional file 1: Fig. 1. Tagln2−/− cDC1s do not optimally control B16F10 tumor growth in mice. (A) Flt3L‑induced cDCs were generated from WT or Tagln2−/− BM cells, and the expressions of the indicated surface markers were analyzed by flow cytometer. (B) Expression of transgelin‑2 in Flt3L‑induced cDC1s. (C) Gross images of OVA+B16F10 solid tumors. C57BL/6 mice were injected i.v. with media alone, WT cDC1s, or Tagln2−/− cDC1s. After 7 days, the mice were s.c. injected with OVA+B16F10 cells. The tumor weights and sizes were quantified at day 8 post tumor inoculation. All data represent the mean of three experiments ± SEM. *P < 0.01. Fig. 2. Recombinant dU‑TG2P potentiated cDC1‑medi‑ated tumor therapy. (A) OTII CD8+ T cells were co‑incubated with none‑ or dU‑TG2P‑treated pOVA (257–264)‑pulsed cDC1s. After 2 h, the cells were then subjected for conjugation assay. (B) After 24 h, culture supernatants from (A) were subjected for cytokine production. (C) After 3 days, T cell proliferation was assessed by Ki‑67/NucSpot double‑positive populations (top) and CTV dilution (bottom). All data represent the mean of three experiments ± SEM. *P < 0.01 (PDF 7390 KB)
Additional file 2. Time‑lapse video showing membrane extensions and cell body movements of WT DCs on Fn‑coated plates in the absence of CCL19 (MP4 2462 KB)
Additional file 3. Time‑lapse video showing membrane extensions and cell body movements of Tagln2−/− DCs on Fn‑coated plates in the absence of CCL19 (MP4 1233 KB)
Additional file 4. Time‑lapse video showing cell spreading, membrane extensions, and cell body movements of WT DCs on Fn‑coated plates in the presence of CCL19 (MP4 2539 KB)
Additional file 5. Time‑lapse video showing cell spreading, membrane extensions, and cell body movements of Tagln2−/− DCs on Fn‑coated plates in the presence of CCL19 (MP4 1032 KB)
Authors’ contributionsH‑RK and C‑DJ conceived the study. H‑RK and J‑SP designed and performed the experiments and analyzed the data. J‑HP, FY, C‑HK, and SKO performed the experiments. I‑JC analyzed the data. H‑RK and C‑DJ wrote and finalized the manuscript. All authors revised the manuscript.
FundingThis work was supported by the Creative Research Initiative Program (2015R1A3A2066253) and the Bio & Medical Technology Development Program (2020M3A9G3080281) through National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (MSIT), the Basic Science Program (2019R1C1C1009570) through National Research Foundation (NRF) grants funded by the Ministry of Education (MOE), the National R&D Program for Cancer Control, Ministry for Health and Welfare (1911264), and supported by GIST Research Institute (GRI) IBBR grant funded by the GIST (2021) and the Joint Research Project of Institutes of Science and Technology (2020–2021), Korea.
Availability of data and materialsAll data generated or analyzed during this study are included in this published article [and its additional files].
Declarations
Ethics approval and consent to participateNot applicable.
Consent for publicationNot applicable.
Competing interestsThe authors have no financial conflicts of interest.
Page 22 of 23Kim et al. J Hematol Oncol (2021) 14:43
Author details1 School of Life Sciences, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi‑ro, Gwangju 61005, Korea. 2 Immune Synapse and Cell Therapy Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Korea. 3 Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609, Rochester, NY 14642, USA. 4 KYNOGEN Co., Suwon 16229, Korea. 5 Department of Hematology‑Oncology, Immunotherapy Innovation Center, Chonnam National University Medical School, Hwasun 58128, Korea.
Received: 11 October 2020 Accepted: 5 March 2021
References 1. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immu‑
nobiology of dendritic cells. Annu Rev Immunol. 2000;18(6):767–811. 2. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic
cells. Cell. 2001;106(3):263–6. 3. Trombetta ES, Mellman I. Cell biology of antigen processing in vitro and
in vivo. Annu Rev Immunol. 2005;23(1):975–1028. 4. Alvarez D, Vollmann EH, von Andrian UH. Mechanisms and consequences
of dendritic cell migration. Immunity. 2008;29(3):325–42. 5. West MA, Prescott AR, Eskelinen EL, Ridley AJ, Watts C. Rac is required for
constitutive macropinocytosis by dendritic cells but does not control its downregulation. Curr Biol. 2000;10(14):839–48.
6. Nobes C, Marsh M. Dendritic cells: New roles for Cdc42 and Rac in anti‑gen uptake? Curr Biol. 2000;10(20):739–41.
7. Tanizaki H, Egawa G, Inaba K, Honda T, Nakajima S, Moniaga CS, et al. Rho‑mDial pathway is required for adhesion, migration, and T‑cell stimulation in dendritic cells. Blood. 2010;116(26):5875–84.
8. Snapper SB, Meelu P, Nguyen D, Stockton BM, Bozza P, Alt FW, et al. WASP deficiency leads to global defects of directed leukocyte migration in vitro and in vivo. J Leukoc Biol. 2005;77(June):993–8.
9. Calle Y, Chou HC, Thrasher AJ, Jones GE. Wiskott‑Aldrich syndrome protein and the cytoskeletal dynamics of dendritic cells. J Pathol. 2004;204(4):460–9.
10. Bouma G, Burns S, Thrasher AJ. Impaired T‑cell priming in vivo resulting from dysfunction of WASp‑deficient dendritic cells. Blood. 2007;110(13):4278–84.
11. Klos Dehring DA, Clarke F, Ricart BG, Huang Y, Gomez TS, Williamson EK, et al. Hematopoietic lineage cell‑specific protein 1 functions in concert with the Wiskott–Aldrich syndrome protein to promote podo‑some array organization and chemotaxis in dendritic cells. J Immunol. 2011;186(8):4805–18.
12. Huang Y, Biswas C, Klos Dehring DA, Sriram U, Williamson EK, Li S, et al. The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells. J Immunol. 2011;187(11):5952–63.
13. Shapland C, Hsuan JJ, Totty NF, Lawson D. Purification and properties of transgelin: a transformation and shape change sensitive actin‑gelling protein. J Cell Biol. 1993;121(5):1065–73.
14. Meng T, Liu L, Hao R, Chen S, Dong Y. Transgelin‑2: A potential oncogenic factor. Tumor Biol. 2017;39(6):101042831770265.
15. Jin H, Cheng X, Pei Y, Fu J, Lyu Z, Peng H, et al. Identification and verification of transgelin‑2 as a potential biomarker of tumor‑derived lung‑cancer endothelial cells by comparative proteomics. J Proteomics. 2016;136:77–88.
16. Na BR, Kim HR, Piragyte I, Oh HM, Kwon MS, Akber U, et al. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. J Cell Biol. 2015;209(1):143–62.
17. Jeon B‑N, Kim H‑R, Chung YS, Na B‑R, Park H, Hong C, et al. Actin stabilizer TAGLN2 potentiates adoptive T cell therapy by boosting the inside‑out costimulation via lymphocyte function‑associated antigen‑1. Oncoim‑munology. 2018;7(12):e1500674.
18. Kim H‑R, Kwon M‑S, Lee S, Mun Y, Lee K‑S, Kim C‑H, et al. TAGLN2 polym‑erizes G‑actin in a low‑ionic state but blocks Arp2/3‑nucleated actin branching in a physiologic condition. Sci Rep. 2018;8(1):5503–18.
19. Kim H‑R, Lee H‑S, Lee K‑S, Jung ID, Kwon M‑S, Kim C‑H, et al. An essential role for TAGLN2 in phagocytosis of lipopolysaccharide‑activated mac‑rophages. Sci Rep. 2017;7(1):8731–44.
20. Yamashiro S. Functions of fascin in dendritic cells. Crit Rev Immunol. 2012;32(1):11–22.
21. Al‑Alwan MM, Rowden G, Lee TDG, West KA. Fascin is involved in the antigen presentation activity of mature dendritic cells. J Immunol. 2001;166(1):338–45.
22. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245–52.
23. Jo S, Kim HR, Mun YV, Jun CD. Transgelin‑2 in immunity: Its implication in cell therapy. J Leukoc Biol. 2018;104(5):903–10.
24. Bachem A, Güttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, et al. Superior antigen cross‑presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells. J Exp Med. 2010;207(6):1273–81.
25. Crozat K, Guiton R, Contreras V, Feuillet V, Dutertre CA, Ventre E, et al. The XC chemokine receptor 1 is a conserved selective marker of mam‑malian cells homologous to mouse CD8α+ dendritic cells. J Exp Med. 2010;207(6):1283–92.
26. Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X, Angel CE, et al. Human CD141+ (BDCA‑3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross‑presents necrotic cell antigens. J Exp Med. 2010;207(6):1247–60.
27. West MA, Prescott AR, Kui MC, Zhou Z, Rose‑John S, Scheller J, et al. TLR ligand‑induced podosome disassembly in dendritic cells is ADAM17 dependent. J Cell Biol. 2008;182(5):993–1005.
28. Tagami H, Aiba S, Nakagawa S, Ozawa H, Miyake K, Yagita H. Up‑regu‑lation of α4 integrin on activated langerhans cells: analysis of adhesion molecules on Langerhans cells relating to their migration from skin to draining lymph nodes. J Invest Dermatol. 1993;100(2):143–7.
29. Hemler ME. VlA proteins in the integrin family: Structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365–400.
30. Sánchez‑Sánchez N, Riol‑Blanco L, Rodríguez‑Fernández JL. The multiple personalities of the chemokine receptor CCR7 in dendritic cells. J Immu‑nol. 2006;176(9):5153–9.
31. Förster R, Davalos‑Misslitz AC, Rot A. CCR7 and its ligands: Balancing immunity and tolerance. Nat Rev Immunol. 2008;8(5):362–71.
32. Kobayashi D, Endo M, Ochi H, Hojo H, Miyasaka M, Hayasaka H. Regula‑tion of CCR7‑dependent cell migration through CCR7 homodimer forma‑tion. Sci Rep. 2017;7(1):8536–50.
33. Akimov V, Barrio‑Hernandez I, Hansen SVF, Hallenborg P, Pedersen AK, Bekker‑Jensen DB, et al. Ubisite approach for comprehensive map‑ping of lysine and N‑terminal ubiquitination sites. Nat Struct Mol Biol. 2018;25(7):631–40.
34. Al‑Alwan MM, Rowden G, Lee TDG, West KA. The dendritic cell cytoskele‑ton is critical for the formation of the immunological synapse. J Immunol. 2001;166(3):1452–6.
35. Reina M, Espel E. Role of LFA‑1 and ICAM‑1 in cancer. Cancers (Basel). 2017;9(11):153.
36. Lu W, Dong Z, Donawho C, Fidler IJ. ICAM‑1 expression and the soluble ICAM‑1 level for evaluating the metastatic potential of gastric cancer. Int J Cancer. 2002;100(4):486–90.
37. Schroder C, Witzel I, Muller V, Krenkel S, Wirtz RM, Janicke F, et al. Prognos‑tic value of intercellular adhesion molecule (ICAM)‑1 expression in breast cancer. J Cancer Res Clin Oncol. 2011;137(8):1193–201.
38. Yoshida A, Okamoto N, Tozawa‑Ono A, Koizumi H, Kiguchi K, Ishizuka B, et al. Proteomic analysis of differential protein expression by brain metas‑tases of gynecological malignancies. Hum Cell. 2013;26(2):56–66.
39. Na BR, Jun CD. TAGLN2‑mediated actin stabilization at the immunological synapse: implication for cytotoxic T cell control of target cells. BMB Rep. 2015;48(7):369–70.
40. Yin LM, Ulloa L, Yang YQ. Transgelin‑2: biochemical and clinical implica‑tions in cancer and asthma. Trends Biochem Sci. 2019;44(10):885–96.
41. Fisher PJ, Bulur PA, Vuk‑Pavlovic S, Prendergast FG, Dietz AB, Doc SVC, et al. Dendritic cell microvilli—a novel membrane structure associated with the multifocal synapse and T cell clustering. Blood. 2008;112(13):5037–46.
42. Brossard C, Feuillet V, Schmitt A, Randriamampita C, Romao M, Raposo G, et al. Multifocal structure of the T cell—dendritic cell synapse. Eur J Immunol. 2005;35(6):1741–53.
Page 23 of 23Kim et al. J Hematol Oncol (2021) 14:43
• fast, convenient online submission
•
thorough peer review by experienced researchers in your field
• rapid publication on acceptance
• support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year •
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your researchReady to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
43. Kim H, Mun Y, Lee K, Park Y, Park J, Park J, et al. T cell microvilli constitute immunological synaptosomes that carry messages to antigen‑presenting cells. Nat Commun. 2018;2018(9):3630–48.
44. Jung Y, Riven I, Feigelson SW, Kartvelishvily E, Tohya K, Miyasaka M, et al. Three‑dimensional localization of T‑cell receptors in relation to microvilli using a combination of superresolution microscopies. Proc Natl Acad Sci. 2016;113(40):E5916–24.
45. Perez CR, De Palma M. Engineering dendritic cell vaccines to improve cancer immunotherapy. Nat Commun. 2019;10(1):5408–17.
Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‑lished maps and institutional affiliations.
![Page 1: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/1.jpg)
![Page 2: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/2.jpg)
![Page 3: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/3.jpg)
![Page 4: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/4.jpg)
![Page 5: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/5.jpg)
![Page 6: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/6.jpg)
![Page 7: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/7.jpg)
![Page 8: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/8.jpg)
![Page 9: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/9.jpg)
![Page 10: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/10.jpg)
![Page 11: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/11.jpg)
![Page 12: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/12.jpg)
![Page 13: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/13.jpg)
![Page 14: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/14.jpg)
![Page 15: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/15.jpg)
![Page 16: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/16.jpg)
![Page 17: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/17.jpg)
![Page 18: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/18.jpg)
![Page 19: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/19.jpg)
![Page 20: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/20.jpg)
![Page 21: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/21.jpg)
![Page 22: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/22.jpg)
![Page 23: Cell-permeable transgelin-2 as a potent therapeutic for dendritic … · 2021. 3. 17. · between DCs and T cells [6]. e formin mDia1 is 5, essential for DC adhesion, migration, and](https://reader036.documents.pub/reader036/viewer/2022071512/61317e1b1ecc51586944c514/html5/thumbnails/23.jpg)
